Thrombin, a multi-functional enzyme already known for its blood-clotting activity, has been recently reported to be an important cell-growth factor. For example, thrombin has been shown to promote angiogenesis, the development of new blood vessels, and to stimulate endothelial cell proliferation. These processes are a pivotal part of healing wounds.
Thrombin peptide derivatives are molecules having an amino acid sequence derived at least in part from that of thrombin, and which are active toward certain thrombin receptors. For example, thrombin peptide derivatives from amino acids 508-530 of human pro-thrombin have been described by the present inventors for promoting thrombin receptor mediated cell stimulation and for their use in the treatment of wounds, and stimulation of angiogenesis (see, e.g., U.S. Pat. No. 5,500,412 or 5,352,664, the contents of which are incorporated herein by reference in their entirety). Because of their biological activity, these thrombin peptide derivatives show great potential as pharmaceuticals. TP508 is one such example of a thrombin peptide derivative and has the amino acid sequence of H-Ala-Gly-Tyr-Lys-Pro-Asp -Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH2 (SEQ ID NO.1).
Strict regulations by the Food and Drug Administration (FDA) require a high degree of purity of biologically active agents when used as pharmaceuticals. It therefore is necessary to obtain active thrombin peptide derivatives that maintain their purity over extended time periods, if these compounds are to be used to treat humans. Unfortunately, the purity of thrombin peptide derivative TP508 diminishes over time because of dimerization resulting from disulfide bond formation. For example, TP508 has a half-life of about 2 to about 4 hours in buffered solutions at neutral pH. Peptide dimers are degradation products and therefore may be considered contaminants of a pharmaceutical composition.
Therefore, there is a need for new peptides with the activity of thrombin peptide derivatives, but which do not form dimers in solution.